Bacteriocins produced by Gram-positive bacteria can be defined as inhibitory substances having a bactericidal mode of action and an essential protein moiety (Jack et al., 1995, Microbiol. Rev. 59:171-200). Bacteriocins produced by Streptococcus mutans were termed mutacins by Hamada and Ooshima (Hamada et al, 1975, Arch. Oral Biol. 20:641-648). Although there are many reports which show that S. mutans produces inhibitory substances, only a few such inhibitors have been isolated and characterized as mutacins (Chikindes et al., 1995, Antimicrob. Agents Chemother. 39:2656-2660; Delisle, 1986, Microbios. 46:21-28; Fukushima et al., 1985, Arch. Oral Biol. 30:229-234; Hamada et al., 1986 Zentralbl. Bakteriol. Hyg. A. 261:287-298; Hillman et al., 1984, Infect. Immun. 44:141-144; Kurita et al., 1988, J. Gen. Microbiol. 134:213-200; Loyola-Rodriguez et al., 1992, J. Gen. Microbiol. 135:269-274; Novak et al., 1994, J. Bacteriol. 176:4316-4320; Novak et al., 1996, Anal. Biochem. 236:358-360; Paul et al., 1975, Infect. Immun. 12:1375-1385; Takada et al., 1984, Infect. Immun. 44:370-378). The well studied mutacins include: RM-10 (Fukushima et al., 1985, Arch. Oral Biol. 3:229-234) C3603 (Takada at al., 1984, Infect. Immun. 44:370-378), JH 1000 (Hillman et al., 1984, Infect. Immun. 44:141-144), GS-5 (Paul et al, 1975, Infect. Immun. 12:1375-1385), MT 3791 and MT 6223 (Hamada et al. 1986, Zentralbl. Bakteriol. Hyg. A. 261:287-298; Loyola-Rodriguez et al., 1992, J. Gen. Microbiol. 138:269-274), mutacin-b (Delisle, 1986, Microbiol. 46:21-28), mutalipocins (Kurita et al., 1988, J. Gen. Microbiol. 134:213-220) and J-T8 (Chikindas et al., 1995, Antimicrob. Agents Chemother. 39:2656-2660; Novak et al., 1994, J. Bacteriol. 176:4316-4320; Novak et al., 1996, Anal. Biochem. 236:358-360).
A preliminary classification of mutacins has been published previously (Morency et al., 1995, Can. J. Microbiol. 41:826-831), but a definitive classification will have to await the complete chemical characterization of these substances. Among the known mutacins, only TB, which belongs to group J (Morency et al., 1995, Can. J. Microbiol. 41:826-831), was identified as a lantibiotic and its first 8 N-terminal amino acid residues were determined by Novak et al. (Novak et al., 1994, J. Bacteriol. 176:4316-4320). Lantibiotics are defined as bacterium-derived ribosomally synthesized lanthionine-containing peptides with antibiotic activity (Jack et al., 1995, Microbiol. Rev. 59:171-200; Bierbaum et al., 1993, Zentralbl. Bakteriol. 278:1-22; Jack et al., 1995, Trends Biotechnol. 13:269-278). They generally contain unsaturated amino acids like 2,3-didehydroalanine (dhA or U) (2)-2,3-didehydrobutyrine (dhB or O), and 2-aminobutyric acid (Abu). The lantibiotics are divided into two types (Jack et al., 1995, Microbiol. Rev. 59:171-200; Bierbaum et al., 1993, Zentralbl. Bakteriol. 278:1-22; Jack et al., 1995, Trends Biotechnol. 13:269-278; Jung, 1991, in: Nisin and Novel Lantibiotics, Jung et al, ads, pp. 1-34 ESCOM Science, Laiden). Type A comprises screw-shaped, amphipathic molecules with molecular masses between 2151 and 4635 Da and with 2 to 7 net positive charges. Type B consists of more globular molecules with molecular masses between 1825 and 2042 Da and with either no net charge or a net negative charge. They usually contain a higher proportion of modified amino acid residues than type A.
Only four streptococcal strains were shown to produce a lantibiotic: Streptococcus pyogenes producing streptococcin A-FF22 (Jack et al., 1994, Eur J Biochem 220:455-462), Streptococcus salivarius producing salivaricin A (Ross et al., 1993, Appl. Environ. Microbiol. 59:2014-2021), Streptococcus mutans TB producing mutacin J-T8 (Parrot et al., 1990, Can. J. Microbiol. 36:123-130; Parrot et al., 1992, Congres de l'AMQ, p. 22; Novak et al., 1993, ASM meeting Abstr. No. A-44, p. 8; Novak et al., 1994, J. Bacteriol 196:4316-4320).
Streptococcus mutans strain B-Ny266 has been previously shown to inhibit 98% of the S. mutans strains tested (Morency et al., 1995, Can. J. Microbiol. 41:826-831) and all tested Gram-positive bacteria including Listeria monocytogenes, Clostridium sporogenes, Mycobacterium phlel, Staphylococcus aureus, Enterococcus faecalis and Bacillus subtilis (Parrot et al., 1990, Can. J. Microbiol. 36:123-130). A preliminary characterization of the antibacterial effect of this strain permitted to hypothesize that at least one mutacin was responsible therefor. However, the mutacin(s) of B-Ny266 has yet to be purified. Furthermore, it remains to be determined whether the wide antibacterial activity associated with strain B-Ny266 is attributable to a single proteinaceous substance or shared by two or more proteinaceous substances therein (Morency et al., 1995, Can. J. Microbiol. 41:826-831; and references therein). It follows, that the determination of the amino sequence of the antibacterial substance(s) present in B-Ny266 had yet to be carried out.
There thus remains a need to purify to homogeneity the bacterial substance(s) carrying the demonstrated antibacterial activity present in S. mutans strain B-Ny266. There further remains a need to identify the amino acid sequence of this substance.
The present invention seeks to meet these and other needs.
The present description refers to a number of documents, the content of which is herein incorporated by reference.